RNA Interference and Heterochromatic Silencing in Replication and Quiescence

通过RNA干扰进行的基因调控通常归因于microRNA，但RNAi具有更古老的 在异染色质沉默和基因组稳定性中起重要作用。异染色质包括 真核染色体的浓缩重复区域，并介导转录沉默， 染色体分离和基因组完整性。我们发现异染色质出乎意料地 转录，随后的RNAi指导组蛋白修饰。在裂殖酵母S. pombe，“co- 转录”沉默发生在细胞周期的S期，随后是转录沉默 然后RNA聚合酶II（Pol II）在复制过程中的释放可以防止DNA损伤，但不 RNAi，复制叉停滞并通过同源重组（HR）修复，导致基因组 不稳定我们已经发现RNAi通过R-loops、RNA-DNA杂合等途径调控基因组的稳定性 在转录复制碰撞，促进HR的结构。 沉默依赖于组蛋白修饰，但最近的研究表明，这可能是一个过于简单化。 首先，组蛋白甲基化招募异染色质蛋白1，其介导液-液相 分离（LLPS），并可能限制对Pol II的访问。第二，RNAi还招募泛素连接酶， 最近发现泛素促进了这些相变。RNAi在C. 线虫和果蝇，但在哺乳动物系统中的保护一直存在争议。例如，在一个示例中， piRNA介导种系中的组蛋白修饰，但不依赖于Dicer，而基因组 Dicer突变小鼠胚胎干细胞（mESC）的不稳定性依赖于卫星转录 重复。引人注目的是，我们发现Dicer-/- mESC中基因组的不稳定性依赖于转录共表达。 激活剂BRD 4和相同的框内布罗莫结构域缺失拯救了裂殖酵母中的Dicer突变体。 S. pombe是细胞周期研究的杰出模型系统，我们是第一个证明， RNAi对于静止（G 0）是必需的。遗传筛选显示，核仁RNA沉默和 组蛋白修饰介导了这一新功能。我们未来五年的目标是确定 RNAi引导异染色质的每个方面从重复不稳定性， 沉默、染色体分离和DNA修复以及在静止中存活。我们最近的工作 表明长的非编码RNA，R环和RNA酶H在上游的活性的中心作用。 在这条路上的步骤。下游事件包括组蛋白修饰和LLPS，它们可能是导致细胞凋亡的基础。 异染色质的典型“浓缩”特性。我们将使用我们的干细胞模型来评估 这些机制在健康和疾病中的保守性和相关性，特别是在癌症中。

Gene regulation by RNA interference is usually attributed to microRNA, but RNAi has a more ancient and fundamental role in heterochromatic silencing and genome stability. Heterochromatin comprises condensed repetitive regions of eukaryotic chromosomes, and mediates transcriptional silencing, chromosome segregation and genome integrity. We have found that heterochromatin is unexpectedly transcribed, and that subsequent RNAi guides histone modification. In the fission yeast S. pombe, “co- transcriptional” silencing occurs during the S phase of the cell cycle, followed by transcriptional silencing thereafter. Release of RNA polymerase II (Pol II) during replication prevents DNA damage, but without RNAi, replication forks stall and are repaired by homologous recombination (HR), causing genome instability. We have found that RNAi regulates genome stability through R-loops, RNA-DNA hybrid structures at transcription-replication collisions that promote HR. Silencing depends on histone modification, but recent studies show this may be an oversimplification. First, histone methylation recruits Heterochromatin Protein 1, which mediates liquid-liquid phase separation (LLPS) and may limit access to Pol II. Second, RNAi also recruits ubiquitin ligase, and we recently found that ubiquitin promotes these phase transitions. RNAi guides histone modification in C. elegans and Drosophila, but conservation in mammalian systems has been controversial. For example, piRNAs mediate histone modification in the germline but do not depend on Dicer, while genome instability in Dicer mutant mouse embryonic stem cells (mESC) depends on transcription of satellite repeats. Strikingly, we have found genome instability in Dicer-/- mESC depends on the transcriptional co- activator BRD4, and identical in-frame bromodomain deletions rescue Dicer mutants in fission yeast. S. pombe is an outstanding model system for cell cycle research, and we were the first to show that RNAi is essential for quiescence (G0). Genetic screens have revealed that nucleolar RNA silencing and histone modifications mediate this novel function. Our goals in the next five years are to determine the elusive mechanism by which RNAi guides each aspect of heterochromatin from repeat instability, to silencing, chromosome segregation and DNA repair as well as survival in quiescence. Our recent work suggests a central role for long non-coding RNA, R-loops and the activity of RNAse H in the upstream steps in this pathway. Downstream events include histone modification and LLPS that may underlie the classically “condensed” properties of heterochromatin. We will use our stem cell model to assess the conservation and relevance of these mechanisms in health and disease, especially in cancer.